Proc. Natl. Acad. Sci. USA Vol. 96, pp. 3217–3222, March 1999 Neurobiology

Cortical bitufted, horizontal, and Martinotti cells preferentially express and secrete into perineuronal nets, nonsynaptically modulating gene expression (GABAergic interneurons͞Ca2؉-binding proteins͞neuropeptides͞chandelier cells͞basket cells)

CHRISTINE PESOLD*†,WEN SHENG LIU*, ALESSANDRO GUIDOTTI*, ERMINIO COSTA*, AND HECTOR J. CARUNCHO*‡

*Psychiatric Institute, Department of Psychiatry, College of Medicine, University of Illinois at Chicago, 1601 West Taylor Street, M͞C 912, Chicago IL, 60612; and ‡Department of Fundamental Biology, University of , Galicia, 15706

Contributed by Erminio Costa, December 28, 1998

ABSTRACT Reelin (Reln) is a protein with some struc- mouse, shows a normal Reln expression in Cajal–Retzius cells tural analogies with other extracellular matrix proteins that and secretion in the extracellular matrix in embryonic devel- functions in the regulation of neuronal migration during the opment but neuroanatomical abnormalities reminiscent of development of cortical laminated structures. In the cortex of those in the reeler mouse (4, 5). It is currently believed that the adult animals, Reln is expressed primarily in ␥-aminobutyric protein encoded by this gene acts downstream of Reln in a acid (GABA)ergic and is secreted into perineuronal signaling pathway that controls laminar corticogenesis and the nets. However, only 50–60% of GABAergic interneurons ex- hippocampal and cerebellar development in mammalian brain press Reln. We have characterized this subpopulation of (6). cortical GABAergic neurons that expresses Reln by using two In adult telencephalon, Reln is preferentially expressed in strategies: (i) a double immunolabeling procedure to deter- GABAergic neurons (7, 8), and Dab1 is expressed predomi- mine the colocalization of Reln with neuropeptides and Ca2؉- nantly in pyramidal cells, as well as in a small population of binding proteins and (ii) a combination of Golgi staining and non-pyramidal cells (9). Indeed, there is a complementary Reln immunolabeling to determine the morphology of the rat pattern in the distribution of Reln and Dab1 in the brain that cortical cells that store Reln. Many interneurons that express suggests a functional link between these two proteins in the Neuropeptide Y (NPY) or somatostatin (but none of those that signaling pathway triggered extracellularly by Reln in adult express parvalbumin) are Reln-immunopositive. A small pop- mammalian brain. ulation of calbindin-positive interneurons and very few cal- Before addressing the functional significance of the pathway retinin-positive cells express Reln immunopositivity. Golgi triggered by Reln extracellularly in adult brain, it is important staining revealed that layer I horizontal cells, layer II–V to consider that perineuronal nets, which include extracellular bitufted neurons, and some deep cortical layer Martinotti matrix proteins (10, 11), may communicate with neurons via cells express Reln. Basket and chandelier cells are often substrate adhesion molecules and integrins (for a review, see immunopositive to parvalbumin, but never to Reln. Although ref. 12). Hence, although the binding site of the released Reln Reln is secreted by GABAergic neurons, its target are not the may be extracellular in the perineuronal nets, it may influence GABA receptors, but rather may be extrasynaptically located neuronal trophic activity indirectly via an interaction with the in perineuronal nets and concerned with the modulation of Dab1 protein. This interaction requires phosphorylation, neuronal plasticity. Dab1, the target adapter protein that which allows this protein to act as an adapter protein operative presumably mediates transcription regulation via the extra- in the translocation of Src kinases from the cytosol to the synaptic actions of Reln, is expressed predominantly in pyra- nucleus (13). Because a high percentage of Reln in the adult midal neurons, but it can also be detected in a small popu- is located in GABAergic interneurons, whose lation of GABAergic neurons that are neither horizontal nor function in the various subtypes differs in a manner related to bitufted neurons. their morphology, it is important to characterize whether a morphologically specialized set of these interneurons ex- The notation ‘‘reelin’’ (Reln) refers to a protein secreted into presses Reln. In an effort to further characterize which sub- the extracellular matrix during embryonic development that is types of GABAergic interneurons express Reln in the adult structurally related to other extracellular matrix proteins (1). cortex, we have used a combination of immunocytochemical In early development, Reln is selectively synthesized and and Golgi-staining techniques. This study will allow us to secreted by specialized neurons (termed Cajal–Retzius) lo- suggest a possible interaction of Reln–Dab1 among specific cated in the marginal zone of the developing brain (1). A populations of cortical GABAergic interneurons, or among mutant mouse that fails to synthesize or secrete this protein these interneurons and pyramidal neurons. This work will also shows a characteristic reeling gate and a disruption of the allow us to address the functional role of Reln released by typical laminated pattern of telencephalic cortex (for review, GABAergic neurons into the extracellular matrix of perineu- see refs. 2 and 3). In these mice, the structure of the hip- ronal nets (10, 11). pocampus and cerebellum is also disrupted, and the cerebellar size is grossly reduced. Another protein, which in embryos is MATERIALS AND METHODS normally expressed by the migrating cortical and hippocampal neurons and cerebellar Purkinje cells, is encoded by a gene A total of 12 adult Fisher rats (180–240 g) were used in this termed Dab1, which is mutated in the scrambler mouse (4–6). study. They were anaesthetized with equithesin and perfused intracardially with saline followed by a cold solution of 4% This neurological phenotype, which is similar to the reeler paraformaldehyde in PBS (pH 7.4). Brains were removed, left

The publication costs of this article were defrayed in part by page charge Abbreviations: GABA, ␥-aminobutyric acid; NPY, neuropeptide Y; payment. This article must therefore be hereby marked ‘‘advertisement’’ in GAD, glutamic acid decarboxylase. accordance with 18 U.S.C. §1734 solely to indicate this fact. †To whom reprint requests should be addressed. e-mail: cpesold@ PNAS is available online at www.pnas.org. psych.uic.edu.

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in fresh fixative for 24 hours at 4°C, and washed in PBS. Brains because of space limitations) were immunostained for Reln to be processed for light microscopy immunolabeling were according to a previously described protocol (7). After dia- infiltrated in 30% sucrose, and 20-␮m thick sections were minobenzidine immunostaining, 80-␮m sections were embed- obtained with a cryostat. For Golgi staining, blocks of cortex ded in LR-white resin. Semithin sections (0.5–1 ␮m in thick- about 1–2 mm thick were cut with a razor blade. ness) containing the neurons of interest were taken with an Double Immunolabeling for Confocal Microscopy. The co- LKB-Nova Biochem ultramicrotome, mounted on slides, and localization of Reln and either somatostatin, neuropeptide Y examined under the light microscope for Reln-immunoreac- (NPY), or calretinin was determined by using a double im- tivity. Photomicrographs of these same cells, now immunola- munofluorescence approach. Because the antibodies against beled for Reln, were taken and deemed Reln-immunopositive parvalbumin and calbindin were raised in the same species as if their cytoplasm was immunostained. the Reln antibody, and similarly the antibodies against Dab1 It is important to note here that Golgi staining does not and GAD67 were raised in the same species, a combination readily infiltrate the myelin sheaths, therefore in adult immunogold͞immunofluorescence was applied. This proce- brain it is sometimes difficult to trace the axonal arborizations. dure allows the silver shell that amplifies the gold particles to It is therefore necessary to impregnate many cells to make an completely engulf the antigenic site, preventing any cross- accurate identification based on their axonal arborization. reactivity. Quantification of the Coexistence of Neuropeptides and For double immunofluorescent labeling, sections were Proteins in GABAergic Interneurons Epressing Reln. To washed in PBS, blocked for 30 minutes in each of RPMI determine which percentage of frontoparietal cortex cells that 2ϩ medium 1640 (GIBCO), 2% normal serum (Chemicon), and express either a given neuropeptide or a Ca -binding protein 1% BSA (Sigma) in PBS before incubation in a solution also express Reln, three consecutive coronal sections from containing both the G-10 (14) primary antibody for Reln each of three animals were studied by using a Leica TCS-NT (1:1,000) and either rat anti-somatostatin (1:500; Chemicon), laser confocal microscope. For each neurochemical marker rabbit anti-calretinin (1:2,000; Chemicon) or rabbit anti-NPY (e.g., calretinin, calbindin, parvalbumin, NPY, and somatosta- (1:5,000; Sigma). After several washes, sections were incubated tin), every cell that contained that marker in the frontoparietal for 1 hour with an anti-mouse secondary antibody conjugated cortex was identified by using the appropriate filter, which was with fluorescein (1:100; Sigma), and with anti-rat or anti-rabbit then switched to determine whether that same immunopositive secondary antibodies conjugated with rhodamine (1:100; cell also contained Reln immunoreactivity. The number of Chemicon). Sections were then rinsed, mounted on slides, single-labeled (e.g., NPY) and double-labeled (e.g., NPY and dried overnight, mounted with a glycerol͞gelatin mounting Reln) cells were counted in layer I, layers II–III, and layers medium (Sigma) containing 25 mg͞ml diazabicyclo[2.2.2]octane IV–VI of the frontoparietal cortex in both hemispheres of each section. The percentage of colocalization between each neu- (DABCO) antifading agent (Sigma), and coverslipped. ϩ ropeptide or Ca2 -binding protein and Reln was then tabu- For the combined immunogold͞immunofluorescent tech- lated over the three consecutive sections from each of three rat nique, Reln and Dab1 (4) were first labeled by immunogold brains. The variation between animals was minimal, being no following a protocol described (15). After silver intensifica- greater than the variation between the left and right hemi- tion, sections were thoroughly rinsed with distilled H O and 2 spheres of each section. blocked with 2% normal serum (30 min) and 1% BSA (30 Morphological Identification of Reln-Immunopositive Cor- min). Reln-labeled sections were then incubated overnight at tical Neurons. Morphological identification of Golgi- 4°C with either mouse anti-calbindin D-28K (1:200; Sigma) or impregnated cells was based on the following classical descrip- mouse anti-parvalbumin (1:2000; Sigma), and Dab1-labeled tions (for a review, see ref. 17): Horizontal cells of layer I sections were incubated with either rabbit anti-GAD67 generally have elongated cell soma that lie parallel to the pia (1:2,000; Chemicon) or the G-10 antibody for Reln (1:1,000). and have that follow the same direction. Bitufted After several rinses, sections were then incubated in anti- cells, which are only found in layers II–V, have an elongated mouse secondary antibody conjugated with fluorescein (1:100; soma that generally lies perpendicular to the pia, with most of Sigma). Sections were then rinsed, mounted on slides, dried the dendritic processes emerging from the top and bottom of overnight, mounted with antifading agent (see above) and the soma. Basket, chandelier, and dendritic-targeting neurons coverslipped. ͞ are generally multipolar cells that have a large soma, which are For each of the above listed fluorochrome or gold primarily located in layers III–V. They are distinguished by fluorochrome combinations, control sections were processed their axonal arborization: Basket-cell run across to simultaneously in which the primary antibody was replaced pyramidal somata, surrounding them and making with 1% BSA in PBS. synaptic contacts with them. Chandelier cells are so named for Golgi Staining and Reln Immunolabeling. Blocks of tissue their axon terminals, which make morphologically specialized were Golgi-stained and deimpregnated as described (16), and contacts with the initial segment of axons, giving then Reln was immunolabeled. Briefly, tissue blocks were the appearance of candles (18). Dendritic-targeting cells, as immersed in a solution of 2% osmium tetroxide in buffer for their name implies, target the basal dendrites of pyramidal 24 hours, followed by 2–4 days immersion in a 4% solution of cells. Pyramidal cells, which are located in layers II–III, V, and potassium dichromate, and then washed and stored for 24 VI, are easily distinguished by their pyramid-shaped somata, hours in a fresh solution of 0.75% silver nitrate. The tissue which have a large apical that runs perpendicular blocks were then transferred to a graded series of increasing from the cell body in the direction of the pial surface. Stellate glycerol concentration and stored in glycerol at 4°C. cells are relatively small star-shaped cells, predominantly These blocks were embedded in 7% agar and cut at 80 ␮m found in cortical layer IV. In layer VI, the majority of nonspiny using a vibratome. Sections were kept for 30 minutes under an neurons are either fusiform (bitufted), or polygonal͞rounded- illuminated device and studied under the microscope. Golgi- shaped neurons. An important subpopulation of these layer impregnated cells of interest were drawn and photographed at V–VI bitufted and multipolar neurons, which send their axons this time. Sections were then rehydrated, gold-toned, and to layer I where they contact the dendritic tufts of pyramidal deimpregnated by treating the sections with 1% sodium thio- cells, are referred to as Martinotti cells. sulfate at 20°C for 80 minutes. This process removed the silver fill from the entire cell, replacing it with a thin shell of silver RESULTS nitrate surrounding the cell soma and proximal dendrites. Each cell was examined at this step, and only cells whose Coexpression in GABAergic Interneurons of Ca2؉-Binding cytoplasm was devoid of silver fill (pictures not included Proteins or Neuropeptides and Reln. Table 1 lists the per- Downloaded by guest on September 28, 2021 Neurobiology: Pesold et al. Proc. Natl. Acad. Sci. USA 96 (1999) 3219

Table 1. Percentage of Ca2ϩ-binding protein or neuropeptide-immunopositive cells that colocalize with Reln in cortical layers of the frontoparietal cortex Neuronal Layer I, Layer II–III, Layer IV–VI, Total, marker % % % % Calretinin 57 26 0 22 Calbindin — 22 27 25 Parvalbumin — 0 0 0 NPY — 82 89 85 Somatostatin 0 50 65 57 In each section of the frontoparietal cortex studied, every neuronal somata positive for a given neuropeptide or protein was verified for Reln-immunoreactivity. We studied each of three consecutive sections from each rat (see Materials and Methods), and the percentage of somata coexpressing one of the markers under study and Reln was tabulated. FIG. 2. Confocal microscope images showing the colocalization for centage of neuronal somata in the frontoparietal cortex that Reln and neuropeptides in layers II–III of 20-␮m sections of the adult are immunopositive for a particular neuronal marker, as well rat frontoparietal cortex. (Upper) Colocalization of Reln (fluorescein; as for Reln. These data show that a high percentage of green) and NPY (rhodamine; red). (Lower) Colocalization between GABAergic interneurons in the frontoparietal cortex that Reln (fluorescein; green) and somatostatin (rhodamine; red). Over- express either the neuropeptide NPY or somatostatin also lays at right are examples of the colocalization frequency between Reln express Reln. Conversely, a much smaller percentage of the and NPY, or somatostatin reported in Table 1. GABAergic neurons that express the Ca2ϩ-binding protein between Reln and the neuropeptides NPY or somatostatin. calretinin or calbindin also express Reln. However, GABAer- The colocalization between Reln and the neuronal markers is gic interneurons that express parvalbumin never express Reln. limited by the variability of the concentration threshold to The results of these confocal studies in the frontoparietal immunodetect these makers. cortex of adult rats are very similar to those recently reported Morphological Identification of the GABAergic Interneu- by Alcantara and her colleagues in the adult mouse brain (22). rons That Express Reln in the Frontoparietal Cortex. The In addition, this study also revealed that Reln does not analysis of coronal sections of the frontoparietal cortex im- colocalize well with either the neuropeptides cholecystokinin munostained with the G-10 mAb against Reln reveals scat- (CCK) or vasoactive intestinal peptide (VIP). The confocal tered immunoreactive somata in all cortical layers, as well as images in Fig. 1 depict the scarcity of colocalization between 2ϩ a diffuse immunostaining of the neuropil in layer I. The Reln and the two Ca -binding proteins under study, while Fig. labeling in the somata and proximal dendrites appears to be 2 gives an example of the higher frequency of colocalization granulated, very likely indicating a higher level of labeling in the rough endoplasmic reticulum and Golgi apparatus; such localization needs to be independently confirmed by electron microscopy. In layer I, Reln-immunopositive somata appear to be rela- tively abundant, especially considering the low cell-body den- sity characteristic of this layer. The morphology of Reln immunoreactive somata of layer I is that of horizontal neurons (Fig. 3A), although there are also some immunopositive neu- rons with rounded or polygonal somata that are not parallel to the pial surface. Layers II and III also include a high number of Reln-immunoreactive neurons. Three different morpholog- ical subtypes can be appreciated: the most abundant are typical bitufted cells (Fig. 3B), however, there are also some immu- noreactive rounded͞polygonal multipolar neurons and some small pyramidal neurons. Layer IV includes some immunopo- sitive multipolar neurons, possibly dendritic targeting neurons, and additional bitufted cells. In layer V, we observed a few scattered pyramidal cells that showed a slight Reln- immunoreactivity (Fig. 3F). In layer VI, the majority of Reln-immunopositive neurons have either fusiform somata or rounded or polygonal somata, many of which may be tenta- tively identified as Martinotti cells (Fig. 3G). By using this combined Golgi-staining͞Reln-immunolabel- ing technique, we could not detect Reln-immunoreactivity in any of the cells that can be morphologically identified as chandelier (Fig. 3C), basket (Fig. 3D), or stellate (Fig. 3E) FIG. 1. Confocal microscope images showing the double labeling cells. 2ϩ ␮ for Reln and several Ca -binding proteins in layers II–III of 20- m Localization of Dab1 in the Adult Rat Frontoparietal Cor- sections of the adult rat frontoparietal cortex. (Top) The double- tex. The confocal images in Fig. 4 show that Dab1 is not found immunofluorescent labeling for Reln (fluorescein; green) and calreti- nin (rhodamine; red). (Middle) Double immunolabeling of Reln (gold; in cells that contain Reln, but rather is predominantly found red) and parvalbumin (fluorescein; green). (Bottom) The double in pyramidal neurons. However, a colocalization of Dab1 with immunolabeling of Reln (gold; red) and calbindin (fluorescein; green). glutamic acid decarboxylase 67 (GAD67), the synthesizing Overlays (Right) show that Reln and these Ca2ϩ-binding proteins are enzyme for GABA, was also detected, suggesting that Dab1 poorly colocalized in the adult rat frontoparietal cortex. may also be expressed in a subpopulation of GABAergic Downloaded by guest on September 28, 2021 3220 Neurobiology: Pesold et al. Proc. Natl. Acad. Sci. USA 96 (1999)

FIG. 4. Confocal microscope images taken from layer V of the adult rat frontoparietal cortex. (Upper) Double labeling of Dab1 (Gold; red) and Reln (fluorescein isothiocyanate; green). Note that Dab1 is predominantly located in pyramidal cells and is not found in Reln-expressing cells. (Lower) Double labeling of Dab1 (Gold; red) and GAD67 (fluorescein isothiocyanate; green). (Left) Whereas Dab1 is predominantly located in pyramidal cells, Dab1 is also found in a small population of nonpyramidal cells (arrows). (Center) Typical distribution pattern of GAD67 (fluorescein; green), both in GABAer- gic cells (arrows) and in the terminal endings surrounding the exci- tatory pyramidal cells. The overlay of the two images (Right) shows that these small Dab1-positive cells are GABAergic cells (arrows). Note also that there is a small population of pyramidal cells that do not contain Dab1, evidenced by the GAD67-labeled outline of pyramidal cells that are not Dab1-positive.

cells that do not express Reln; this possibility is currently under investigation. However, not all pyramidal neurons contain Dab1, which is evidenced by the outline of GAD67- immunopositive terminals on typical pyramidal cell somata, which are not immunoreactive to the Dab1 antibody (see Fig. 4).

DISCUSSION A previous report has shown that in adult rat cortex, at least 70% of the neurons that express Reln are GAD67- immunopositive (7). The Golgi-staining͞Reln-immunolabel- ing method has allowed us to determine that Reln is expressed preferentially by horizontal cells in layer I, bitufted cells in layers II–V, and presumed Martinotti cells of layer VI. These findings correspond well with the histochemical colocalization studies showing that Reln co-exits with calbindin D-28K, a Ca2ϩ-binding protein reportedly expressed by double bouquet cells (17, 20), as well as with somatostatin, a neuropeptide expressed by Martinotti cells (21). A population of multipolar neurons, as well as a very small population of pyramidal FIG.3. (Left) Camera Lucida drawings of several morphologically neurons, express Reln, but in pyramidal neurons, the Reln identified neurons from the adult rat frontoparietal cortex. (Center) expression level is very low. The Golgi-impregnation studies Photomicrograph of the somata of the cells drawn at Left.(Right) ␮ fail to detect Reln expression in chandelier or basket cells, Photomicrographs of 1- m sections taken through the same cells which is in agreement with the lack of Reln colocalization with following removal of the silver impregnation, gold-toning, which outlines the cell soma with a gold shell, and immunolabeled for Reln. parvalbumin, which is normally expressed in chandelier and (A) Horizontal neuron of layer I. Note the cytoplasm immunostained basket cells (20, 22–25). for Reln (Right). (B) Bitufted neuron of layer III. Reln immunostain- Some authors have postulated that only a few Cajal–Retzius ing is very evident in the cell cytoplasm (Right). (C) Chandelier neuron cells (a neuronal phenotype that produces Reln from embry- of layer IV. Note the unstained cytoplasm indicating a lack of onic day 9 until 10–15 days postnatal) could survive in the D) Basket neuron of layer IV. Lack adult brain and be located either in layer I or could have) .(ء) immunoreactivity for Reln of immunoreactivity for Reln is evidenced by the unstained cytoplasm migrated to deeper layers (26–28). However, it is very likely (F) .(ء) E) Aspiny stellate cell of layer IV. Reln-immunonegative) .(ء) Pyramidal neuron of layer V. A low level of immunoreactivity can be that most of them die by the end of the second postnatal week detected in the cytoplasm. (G) Martinotti cell of layer VI. Stained in the rat (27, 29). Although it cannot be excluded that a few cytoplasm indicates immunoreactivity for Reln. (Bars ϭ 10 ␮m.) Cajal–Retzius cells might survive longer than two weeks postnatally and continue to express Reln into adulthood, the interneurons. These latter neurons do not have the typical majority of Reln-positive GABAergic interneurons expressed elongated somata of the Reln-containing bitufted or horizon- in the adult brain belong to an entirely new generation of tal cells and therefore may be the basket and͞or chandelier neurons that did not express Reln during development. This Downloaded by guest on September 28, 2021 Neurobiology: Pesold et al. Proc. Natl. Acad. Sci. USA 96 (1999) 3221

view in rodents also is supported by the observation that during the first postnatal week, when Cajal–Retzius cells are still abundant, additional cortical neurons expressing Reln are already evident (30). In the cortical marginal zone of humans at about 27 weeks of gestation, after Cajal–Retzius cells disappear, other Reln-expressing neuronal subtypes begin to emerge (31). The Reln secreted by Cajal–Retzius cells and Reln secreted in adult life by other neurons share many structural charac- teristics and physicochemical properties with other extracel- lular matrix protein expressed in perineuronal nets (10, 11). Like these matrix proteins, Reln expresses eight epidermal growth factor-like motifs and is endowed with an N terminus signal peptide that is required for its secretion (1). In a previous study using electron microscopy (7), we have shown that Reln is expressed in cerebellar parallel fibers, suggesting it might be transported from the granule cell bodies to the molecular layer for its release extracellularly into the perineu- ronal nets in the proximity of the Dab1-expressing Purkinje cells and their dendrites (9). Based on this observation, one might infer that Reln released extracellularly from either axon terminals or dendrites of cortical interneurons may be an- chored to the extracellular matrix structure expressed as a component of the perineuronal nets (10, 11) and connected to the dendrites of certain interneurons by substrate-adhesion molecules that link perineuronal nets to integrin receptors (see Fig. 5). It is important to note that Reln-containing GABAergic interneurons do not express the adapter protein Dab1. During embryonic brain development, Dab1 is expressed by neurons that migrate under Reln control (4). In the adult neocortex, Dab1 is primarily expressed in pyramidal neurons and in Purkinje cells and their dendrites (9), as well as in a small population of GABAergic interneurons in the adult rat cortex (Fig. 4). The dendrites of pyramidal cells are the main targets FIG. 5. Reln is released extracellularly from bitufted, horizontal, of GABAergic interneurons that secrete Reln extracellularly. and Martinotti GABAergic cells of the frontoparietal cortex. Because The axons of layer I horizontal cells and Martinotti cells of a Reln-specific recognition site was not found in various GABA layer VI, contact the dendritic tufts of pyramidal neurons in receptor subtypes, the integrin-dependent Reln stimulation of contig- layer I (17). The transport of Reln in axons or dendrites, its uous cells may occur via an RGD motif (Arginine, Glycine, Aspartate) possible extracellular release, and subsequent anchoring to expressed in matrix adhesion proteins (12). Thus, a Reln-dependent intraneuronal cascade of events is triggered, leading to the nuclear specific sites in the extracellular matrix, could account for the translocation of Src-family tyrosine kinases mediated by the adapter diffuse Reln immunolabeling observed in layer I, not only in protein Dab1. This translocation can modulate the expression of rodents (7) but also in humans (9). Bitufted cells project mostly specific patterns of DNA transcription. It is hypothesized that Reln, to dendritic spines and shafts of pyramidal neurons, in both which structurally resembles other extracellular matrix proteins, may superficial and deeper cortical layers (17). In addition, bitufted serve as an important regulatory link to harmonize regulation of cells contact other GABAergic interneurons such as a sub- specific genes to the frequency of columnary pyramidal cell firing. population of chandelier and basket cells, which may be those Those pyramidal or GABAergic neurons, which express Dab1, trans- that express Dab1 (see Fig. 4). And although there is a small duce the signal cascade triggered by extracellular Reln via the p35 population of Reln-containing pyramidal cells, there is also a activator protein of cdk5 (cyclin-dependent kinase 5) and cause binding of a nonreceptor tyrosine kinase (i.e., Fyn-K) to the adapter small population of pyramidal cells that do not contain Dab1 protein modified by phosphorylation (4, 6, 37). It is now being (Fig. 4). Further investigation is required to clarify whether the established beyond dispute that extracellular matrix proteins around pyramidal neurons that are immunonegative for Dab1 are also the neurons are organized in a perineuronal net (10, 11). This net those that are immunopositive for Reln. includes 3 layers: (i) integrin receptors form the layer contiguous with Dab1 is mostly a cytoplasmic protein that may be trans- the intracellular milieu; (ii) the ‘‘matrix proper’’ where Reln is secreted ported in dendrites of pyramidal neurons, as it is transported and interacts with RGD; and (iii) an external layer rich in carbohy- in dendrites of Purkinje cells (7). After Dab1 phosphorylation, drates, such as hyaluronic and sialic acid (12). triggered by Reln anchored to the extracellular matrix (see Fig. 5), Dab1 may act as an adapter protein operative in the of the specific cyclin-dependent kinase 5 (cdk5) activator transport to the nucleus of Src family nonreceptor tyrosine called p35. This in turn could cause the phosphorylation of kinases such as fyn (5, 13). When these proteins reach the Dab1, which is a rate-limiting event that enables the adapter nucleus, they participate in the regulation of protein synthesis function of this protein (13). In support of this speculation is related to neuronal plasticity. It is possible that Reln in bitufted the evidence that knockout mice of Dab1 protein (4), cdk5 cells may be secreted in the vicinity of Dab1-containing (32), and p35 activator (33) can cause a corticogenic defect pyramidal or GABAergic cells, where its extrasynaptic actions similar but not identical to that of the scrambler (34) and reeler may be involved in the regulation of protein synthesis. Ac- (1) mice. cording to the model shown in Fig. 5, extracellular Reln could In summary, the activation of a nonreceptor tyrosine kinase interact with RGD-containing substrate adhesion-promoting by an unknown signaling cascade following integrin activation proteins that may participate in the neuronal internalization of by extracellular matrix adhesion proteins could bring about a the Reln transduction pathway via membrane integrin recep- cascade of events affecting DNA transcription in pyramidal tors. The activation of this receptor may prompt the interaction neurons, and presumably also in cortical GABAergic inter- Downloaded by guest on September 28, 2021 3222 Neurobiology: Pesold et al. Proc. Natl. Acad. Sci. USA 96 (1999)

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